Androgens and masculinization of genitalia in the spotted hyaena (Crocuta crocuta). 1. Urogenital morphology and placental androgen production during fetal life

The pre-T cell receptor (TCR) associates with CD3-transducing subunits and triggers the selective expansion and maturation of T cell precursors expressing a TCR-beta chain. Recent experiments in pre-Talpha chain-deficient mice have suggested that the pre-TCR may not be required for signaling allelic exclusion at the TCR-beta locus. Using CD3-epsilon- and CD3-zeta/eta-deficient mice harboring a productively rearranged TCR-beta transgene, we showed that the CD3-gammadeltaepsilon and CD3-zeta/eta modules, and by inference the pre-TCR/CD3 complex, are each essential for the establishment of allelic exclusion at the endogenous TCR-beta locus. Furthermore, using mutant mice lacking both the CD3-epsilon and CD3-zeta/eta genes, we established that the CD3 gene products are dispensable for the onset of V to (D)J recombination (V, variable; D, diversity; J, joining) at the TCR-beta, TCR-gamma, and TCR-delta loci. Thus, the CD3 components are differentially involved in the sequential events that make the TCR-beta locus first accessible to, and later insulated from, the action of the V(D)J recombinase.     

The CD3gamma chain is essential for development of both the TCRalphabeta and TCRgammadelta lineages

CD3gamma and CD3delta are the most closely related CD3 components, both of which participate in the TCRalphabeta-CD3 complex expressed on mature T cells. Interestingly, however, CD3delta does not appear to participate functionally in the pre-T-cell receptor (TCR) complex that is expressed on immature T cells: disruption of CD3delta gene expression has no effect on the developmental steps controlled by the pre-TCR. Here we report that in contrast with CD3delta, CD3gamma is an essential component of the pre-TCR. We generated mice selectively lacking expression of CD3gamma, in which expression of CD3delta, CD3epsilon, CD3zeta, pTalpha and TCRbeta remained undisturbed. Thus, all components for composing a pre-TCR are available, with the exception of CD3gamma. Nevertheless, T-cell development is severely inhibited in CD3gamma-deficient mice. The number of cells in the thymus is reduced to <1% of that in normal mice, and the large majority of thymocytes lack CD4 and CD8 and are arrested at the CD44-CD25+ double negative (DN) stage of development. Peripheral lymphoid organs are also practically devoid of T cells, with absolute numbers of peripheral T cells reduced to only 2-5% of those in normal mice. Both TCRalphabeta and TCRgammadelta lineages fail to develop effectively in CD3gamma-deficient mice, although absence of CD3gamma has no effect on gene rearrangements of the TCRbeta, delta and gamma loci. Furthermore, absence of CD3gamma results in a severe reduction in the level of TCR and CD3epsilon expression at the cell surface of thymocytes and peripheral T cells. The defect in the DN to double positive transition in mice lacking CD3gamma can be overcome by anti-CD3epsilon-mediated cross-linking. CD3gamma is thus essential for pre-TCR function.     

Proliferation kinetics associated with T cell receptor-beta chain selection of fetal murine thymocytes

After productive rearrangement of a TCR beta chain gene, CD4-8- double negative (DN) thymocytes express TCR beta polypeptide chains on the cell surface together with pre-T alpha and the CD3 complex forming the pre-TCR. Signals transmitted through the pre-TCR select TCR beta + DN thymocytes for further maturation to the CD4+8+ double positive stage, whereas DN cells that fail to generate a productive TCR beta gene rearrangement do not continue in development. This process is termed TCR beta chain selection. Although it is likely that differences between proliferation dynamics of TCR beta + and TCR beta-cells may play a role, the exact mechanisms of TCR beta chain selection have not been elucidated. We therefore studied the proliferation dynamics of TCR beta + and TCR beta-thymocytes during fetal development, i.e., when TCR beta chain selection takes place for the first time. We analyzed in situ accumulation of TCR beta + thymocytes by confocal microscopy, and determined cell cycle and division parameters of TCR beta + and TCR beta-populations by flow cytometry. About 600 TCR beta + cells/thymic lobe are generated by independent induction events between days of gestation (dg) 13.5, and 15.5. As of dg 14.5, most TCR beta + cells have entered S/G2 phase of cell cycle, followed by seven to eight rapid cell divisions in fetal thymic organ culture, suggesting a corresponding burst of nine cell divisions within 4 d in vivo. By dg 18.5, the division rate of TCR beta + cells has slowed down to less than 1/d. About three quarters of TCR beta-cells divide at a slow rate of 1/d on dg 14.5, the proportion of nondividing cells increasing to 50% within the following four d. From dg 16.5 onwards, TCR beta-cells, but not TCR beta + cells, contain a significant proportion of apoptotic cells. The results suggest that failure to become selected results in shutdown of proliferation and eventual programmed cell death of fetal TCR beta-cells. Positive selection of fetal TCR beta + cells is achieved by an increased rate of cell divisions lasting for approximately 4 d.     

CD5 expression is developmentally regulated by T cell receptor (TCR) signals and TCR avidity

Recent data indicate that the cell surface glycoprotein CD5 functions as a negative regulator of T cell receptor (TCR)-mediated signaling. In this study, we examined the regulation of CD5 surface expression during normal thymocyte ontogeny and in mice with developmental and/or signal transduction defects. The results demonstrate that low level expression of CD5 on CD4(-)CD8(-) (double negative, DN) thymocytes is independent of TCR gene rearrangement; however, induction of CD5 surface expression on DN thymocytes requires engagement of the pre-TCR and is dependent upon the activity of p56(lck). At the CD4(+)CD8(+) (double positive, DP) stage, intermediate CD5 levels are maintained by low affinity TCR-major histocompatibility complex (MHC) interactions, and CD5 surface expression is proportional to both the surface level and signaling capacity of the TCR. High-level expression of CD5 on DP and CD4(+) or CD8(+) (single positive, SP) thymocytes is induced by engagement of the alpha/beta-TCR by (positively or negatively) selecting ligands. Significantly, CD5 surface expression on mature SP thymocytes and T cells was found to directly parallel the avidity or signaling intensity of the positively selecting TCR-MHC-ligand interaction. Taken together, these observations suggest that the developmental regulation of CD5 in response to TCR signaling and TCR avidity represents a mechanism for fine tuning of the TCR signaling response.     

Thymocyte development in the absence of pre-T cell receptor extracellular immunoglobulin domains

Immature thymocytes express a pre-T cell receptor (pre-TCR) composed of the TCRbeta chain paired with pre-Talpha. Signals from this receptor are essential for passage of thymocytes through a key developmental checkpoint in the thymus. These signals were efficiently delivered in vivo by a truncated form of the murine pre-TCR that lacked all of its extracellular immunoglobulin domains. De novo expression of the truncated pre-TCR or an intact alphabetaTCR was sufficient to activate characteristic TCR signaling pathways in a T cell line. These findings support the view that recognition of an extracellular ligand is not required for pre-TCR function.     

Preferential requirement of CD3 zeta-mediated signals for development of immature rather than mature thymocytes

Antigen recognition signals by the TCR are transduced through activation motifs present in the cytoplasmic region of CD3 chains. In vitro analysis has suggested that the CD3zeta chain mediates different signals from other CD3 chains. To analyze the in vivo function of CD3zeta-mediated signals for T cell development, mice expressing a mutant CD3zeta chain lacking all the activation motifs were generated by introducing the transgene into zeta-knockout mice. Mature CD4(+) single-positive (SP) thymocytes in these mice were greater in number than in zeta-deficient mice, and the promoted differentiation was indicated by the changes of CD69 and HSA phenotypes. We found that even in the absence of activation motifs in CD3zeta, these mature cells became functional, being able to induce Ca2+ mobilization and proliferation upon stimulation. On the other hand, CD4(-)CD8(-) double-negative (DN) thymocytes, most of which were arrested at the CD44(-)CD25(+) stage similarly to those in zeta-deficient mice, could not be promoted for differentiation into CD4(+)CD8(+) double-positive thymocytes in these mice in spite of the fact that the expression of the transgene in DN thymocytes was higher than that of zeta in wild-type mice. These results demonstrate the preferential dependence of the promotion of development and/or expansion of DN thymocytes rather than mature thymocytes upon the activation signals through the zeta chain and suggest differential requirements of TCR signaling for mature SP and immature DN thymocyte developments in vivo.     

Cell-fate decisions in early T cell development: regulation by cytokine receptors and the pre-TCR

During lymphocyte development, cell-fate decisions are determined by a myriad of signals produced by the micro- environment of the thymus and the bone marrow. These yet to be fully defined developmental cues regulate stage-specific gene expression, and the extraordinarily well-characterized stages of T and B cell development have provided attractive model systems for studying regulation of cellular differentiation. In particular, studies on the contribution of both antigen receptors and cytokine receptors to lymphoid development have illuminated essential signalling pathways in early T and B cells. Here, we review investigations supporting an obligatory role for the IL-7 receptor pathway in early T cell development. IL-7 is produced by both thymus and bone marrow stromal cells, and its potential contribution to survival, differentiation and proliferation of pro-T cells is discussed. We also address the contribution of the pre-T cell receptor (pre-TCR) to differentiation past the pro-T cell stage, and recent advances in deciphering the composition and function of the pre-TCR complex are discussed. Finally, we suggest future directions in this field that may serve to reveal whether and how signals initiated by the cytokine receptors and pre-TCR may intersect, and to define which down-stream molecular events are regulated by these receptors.     

Immature thymocyte antigen-1: a novel thymocyte marker discriminating pre- and post-selected thymocytes

Previously, we described a mAb (1-23) reacting with a novel cell surface antigen expressed on thymocytes at late CD4-CD8- [(double negative (DN)] to early CD4+CD8+ [(double positive (DP)] differentiation stage. Since the expression of this molecule was restricted to immature thymocytes, we designated it as immature thymocyte antigen-1 (IMT-1). In this study, we have investigated the relevance of IMT-1 expression to thymocyte selection using TCR transgenic mice, scid mice or RAG-2-/- mice. The IMT-1+ population in DP thymocytes was decreased in the thymuses of MHC class I-restricted or class II-restricted TCR transgenic mice with a positively selecting MHC background when compared with that of the mice with a non-selecting MHC background. IMT-1+ DP thymocytes were also decreased in TCR transgenic mice in which negative selection occurs. When DP thymocytes in H-Y TCR transgenic mice were stimulated with CD3epsilon mAb in vitro as well as in vivo, the expression of IMT-1 on DP thymocytes was decreased. Furthermore, the expression of IMT-1 on DN thymocytes from RAG-2-/- mice was drastically reduced when CD3epsilon mAb was challenged in vivo. These results suggest that the expression of IMT-1 on DP or DN thymocytes is down-regulated by stimulation through TCR as well as pre-TCR. Taken together, these results show that IMT-1 is a unique surface marker which exquisitely separates pre-selected thymocytes from post-selected thymocytes.     

Calnexin associates with monomeric and oligomeric (disulfide-linked) CD3delta proteins in murine T lymphocytes

The antigen-binding receptor expressed on most T lymphocytes consists of disulfide-linked clonotypic alphabeta heterodimers noncovalently associated with monomeric CD3gamma,delta,epsilon proteins and disulfide-linked zeta zeta homodimers, collectively referred to as the T cell antigen receptor (TCR) complex. Here, we examined and compared the disulfide linkage status of newly synthesized TCR proteins in murine CD4(+)CD8(+) thymocytes and splenic T cells. These studies demonstrate that CD3delta proteins exist as both monomeric and oligomeric (disulfide-linked) species that differentially assemble with CD3epsilon subunits in CD4(+)CD8(+) thymocytes and splenic T cells. Interestingly, unlike previous results on glucose trimming and TCR assembly of CD3delta proteins in splenic T cells (Van Leeuwen, J. E. M., and K. P. Kearse (1996) J. Biol. Chem. 271, 9660-9665), we found that glucose residues were not invariably removed from CD3delta glycoproteins prior to their assembly with CD3epsilon subunits in CD4(+)CD8(+) thymocytes. Finally, these studies show that calnexin associates with both monomeric and disulfide-linked CD3delta proteins in murine T cells. The data in the current report demonstrate that CD3delta proteins exist as both monomeric and disulfide-linked molecules in murine T cells that differentially associate with partner TCR chains in CD4(+)CD8(+) thymocytes and splenic T cells. These results are consistent with the concept that folding and assembly of CD3delta proteins is a function of their oxidation state.     

K+ channel inactivation mediated by the concerted action of the cytoplasmic N- and C-terminal domains

Mice lacking thymic function of the GTPase Rho show severe defects in fetal and adult thymopoiesis. Rho thymi are deficient in CD44+ CD25+ pro-T cells and CD44- CD25+ early pre-T cells because Rho function is required for survival but not G1/S phase cell cycle progression in these populations. The selective apoptosis defect in Rho prothymocytes can be rescued by expression of a bcl-2 transgene. A second function for Rho is seen in CD44- CD25- late pre-T cells: Rho regulates cell cycle progression but not survival of this population. These studies show that the critical processes of proliferation and survival are independently regulated during thymopoiesis and establish two different functions for Rho in the development of early thymic progenitors.     

Defective interactions between TCR chains and CD3 heterodimers prevent membrane expression of TCR-alpha beta in human T cells

The human TCR complex is composed of two clonotypic polypeptide chains, TCR-alpha and TCR-beta (or TCR-gamma and TCR-delta) associated with CD3 gamma-, delta-, and epsilon-chains and zeta 2 homodimers. All six polypeptide chains are indispensable for TCR membrane expression and signaling function. In the present paper is described the analysis of a new TCR membrane-negative Jurkat T cell variant: E6.R3. The defect in this variant bears on the interaction between TCR and CD3 chains. E6.R3 cells have deleted three nucleotides in the TCR-alpha transmembrane (TM) region, which consequently lacks a leucine. This defect causes 1) lack of association between TCR alpha-chains and CD delta epsilon heterodimers; 2) lack of formation of disulphide-linked, fully glycosylated TCR-alpha beta heterodimers; and 3) lack of interaction between TCR-alpha beta/CD3 complexes and zeta-chains. Despite these defective interactions, TCR alpha-chains appear to become fully glycosylated, i.e., they are not retained in the endoplasmic reticulum but are further processed in the Golgi apparatus without such interactions. The defect may be due to the observation that in the E6.R3 TCR alpha- chains TM region, the two charged amino acids are situated on the same side of the alpha-helix; these two amino acids are exposed on opposite faces of the TM alpha-helix in normal TCR alpha-chains, possibly allowing TCR alpha-chains to interact with both CD3 delta- and CD3 epsilon-chains. Further possible consequences of the leucine deletion in the E6.R3 TCR-alpha TM region are discussed.     

Styryl-pyrones from Goniothalamus arvensis

CD3gamma and CD3delta are two highly related components of the T cell receptor (TCR)-CD3 complex which is essential for the assembly and signal transduction of the T cell receptor on mature T cells. In gene knockout mice deficient in either CD3delta or CD3gamma, early thymic development mediated by pre-TCR was either undisturbed or severely blocked, respectively, and small numbers of TCR-alphabeta+ T cells were detected in the periphery of both mice. gammadelta T cell development was either normal in CD3delta-/- mice or partially blocked in CD3gamma-/- mice. To examine the collective role of CD3gamma and CD3delta in the assembly and function of pre-TCR and in the development of gammadelta T cells, we generated a mouse strain with a disruption in both CD3gamma and CD3delta genes (CD3gammadelta-/-). In contrast to mice deficient in either CD3gamma or CD3delta chains, early thymic development mediated by pre-TCR is completely blocked, and TCR-alphabeta+ or TCR-gammadelta+ T cells were absent in the CD3gammadelta-/- mice. Taken together, these studies demonstrated that CD3gamma and CD3delta play an essential, yet partially overlapping, role in the development of both alphabeta and gammadelta T cell lineages.     

Rapid deletion of rearranged T cell antigen receptor (TCR) Valpha-Jalpha segment by secondary rearrangement in the thymus: role of continuous rearrangement of TCR alpha chain gene and positive selection in the T cell repertoire formation

A rearranged T cell receptor (TCR) Valpha and Jalpha gene from a cytochrome c-specific T cell hybridoma was introduced into the genomic Jalpha region. The introduced TCR alpha chain gene is expressed in a majority of CD3 positive and CD4 CD8 double-negative immature thymocytes. However, only a few percent of the double-positive and single-positive thymocytes express this TCR alpha chain. This decrease is caused by a rearrangement of TCR alpha chain locus, which deletes the introduced TCR gene. Analysis of the mice carrying the introduced TCR alpha chain and the transgenic TCR beta chain from the original cytochrome c-specific T cell hybridoma revealed that positive selection efficiently rescues double-positive thymocytes from the loss of the introduced TCR alpha chain gene. In the mice with negatively selecting conditions, T cells expressing the introduced TCR alphabeta chains were deleted at the double-positive stage. However, a large number of thymocytes escape negative selection by using an endogenous TCR alpha chain created by secondary rearrangement maintaining normal thymocyte development. These results suggest that secondary rearrangements of the TCR alpha chain gene play an important role in the formation of the T cell repertoire.     

Rescue of thymocytes from glucocorticoid-induced cell death mediated by CD28/CTLA-4 costimulatory interactions with B7-1/B7-2

During the differentiation of thymocytes to mature T cells the processes of positive and negative selection result in signals that either protect thymocytes from cell death, or delete, through apoptosis, thymocytes with self-reactive T cell receptors (TCR). Glucocorticoids have been shown to induce thymocyte apoptosis and are produced within the thymic microenvironment. Furthermore, steroid-induced apoptosis of thymocytes has been suggested as a potential mechanism for removal of nonselected thymocytes. In this report, we demonstrate that thymocytes can be rescued from glucocorticoid-induced apoptosis by incubation with cells that express high levels of B7-1 or B7-2. In addition, the ability to be rescued by B7-1 and/or B7-2 can precede expression of the TCR. We demonstrate that CD3(+)-depleted or CD3+/ TCR-beta(+)-doubly depleted thymocytes can be rescued from glucocorticoid-induced apoptosis through the interaction of CD28 or CTLA-4 on thymocytes with cells bearing high levels of B7-1 or B7-2. Furthermore, these transfected cells are major histocompatibility complex (MHC) class II negative and, while they may express MHC class I, there is no preferential rescue of CD8+ thymocytes in the presence of glucocorticoids. Together, these data suggest that the rescue of thymocytes from glucocorticoids can be independent of the TCR. We also demonstrate that, in addition to CD28, CTLA-4 is expressed on thymocytes, suggesting that rescue from glucocorticoid-induced cell death can be mediated by both CD28 and CTLA-4. A CTLA-4Ig fusion protein which binds to both B7-1 and B7-2 was shown to completely block the rescue of thymocytes from glucocorticoid-induced cell death. Therefore, we conclude that interactions between B7-1/B7-2 and CD28/CTLA-4 are sufficient and necessary for rescue of thymocytes from glucocorticoid-induced cell death.     

The expression of CD4 by T cell precursors resident in both the thymus and the bone marrow

A very small fraction of thymocytes has recently been identified that expresses low levels of CD4 in the absence of CD8, CD3, or a TCR. These CD4lo thymocytes appear to be the precursors of the early CD4-CD8-CD3- thymic subset and contain most of the T cell progenitor activity found within the thymus. Here, we examined adult bone marrow for the presence of a similar population of cells and found that 0.5% to 3.5% of C58/J bone marrow cells express low, but detectable levels of CD4 (CD4lo) at the cell surface in the absence of CD3. These CD4lo bone marrow cells display pre-T cell activity, in that they are able to repopulate the thymus of irradiated recipient mice after intrathymic transfer. Moreover, we found that most of pre-T cell activity found in the bone marrow is contained within the CD4lo expressing subset of marrow cells. Although the CD4lo cells found in both the thymus and bone marrow display pre-T cell activity, the CD4lo cells from these two sites showed pronounced differences with respect to their ability to respond to specific cytokine stimulation in vitro. Bone marrow-resident CD4lo cells proliferated in response to both IL-3 and mast cell growth factor in vitro, whereas CD4lo cells isolated from the thymus did not. Furthermore, CD4lo bone marrow cells, grown in media containing IL-3 and mast cell growth factor, retained their pre-T cell activity, indicating that CD4lo cells with pre-T cell capabilities were among the IL-3 and mast cell growth factor-responsive cells. These data suggest that although pre-T cells in bone marrow share the CD4lo phenotype with their intrathymic counterparts, they may be fundamentally different with respect to the environmental factors that control their growth.     

Specific requirement for CD3epsilon in T cell development

T cell antigen receptor (TCR) and pre-TCR complexes are composed of clonotypic heterodimers in association with dimers of signal transducing invariant subunits (CD3gamma, -delta, -epsilon, and zeta). The role of individual invariant subunits in T cell development has been investigated by generating gene-specific mutations in mice. Mutation of CD3gamma, -delta, or zeta results in an incomplete block in development, characterized by reduced numbers of mature T cells that express low levels of TCR. In contrast, mature T cells are absent from CD3epsilon-/- mice, and thymocyte development is arrested at the early CD4(-)CD8(-) stage. Although these results suggest that CD3epsilon is essential for pre-TCR and TCR expression/function, their interpretation is complicated by the fact that expression of the CD3gamma and CD3delta genes also is reduced in CD3epsilon-/- mice. Thus, it is unclear whether the phenotype of CD3epsilon-/- mice reflects the collective effects of CD3gamma, -delta, and -epsilon deficiency. By removing the selectable marker (PGK-NEO) from the targeted CD3epsilon gene via Cre/loxP-mediated recombination, we generated mice that lack CD3epsilon yet retain normal expression of the closely linked CD3gamma and CD3delta genes. These (CD3epsilonDelta/Delta) mice exhibited an early arrest in T cell development, similar to that of CD3epsilon-/- mice. Moreover, the developmental defect could be rescued by expression of a CD3epsilon transgene. These results identify an essential role for CD3epsilon in T cell development not shared by the CD3gamma, CD3delta, or zeta-family proteins and provide further evidence that PGK-NEO can influence the expression of genes in its proximity.